Mouse mammary tumor virus (MMTV) genomes stably integrated into the DNA of cultured mammalian cell lines will be used as a model system for studying control of gene expression. The rate of MMTV RNA synthesis is stimulated dramatically and specifically by glucocorticoid hormones. Thus, MMTV provides an excellent experimental system for studying transcriptional regulation and the mechanism of glucocorticoid hormone action. Using (Beta-S)ribonucleoside triphosphates, a cell free transcription system will be established to study initiation of MMTV RNA synthesis. This cell free system will be used to determine whether glucocorticoids control MMTV RNA synthesis at the level of transcription initiation. Nucleic acid hybridization techniques will be developed to characterize MMTV RNA synthesis with regard to the degree of symmetry of transcription and the identity and location of the initiating nucleotide(s). The transcription system will be adapted to carry out and study the entire glucocorticoid response in a cell free system. Immunological selection procedures directed toward MMTV antigens will be developed to obtain cell lines with altered MMTV gene expression and/or Glucocorticoid-mediated regulation. The glucocorticoid receptor proteins and the MMTV proviruses, transcripts, and antigens of these phenotypic variants will be characterized in comparison to wild type cells. Somatic cell hybrids will be constructed to test for phenotypic complementation between different variant lines. Eventually, nuclear and cytoplasmic preparation from variants will be used in the cell free hormone response system as a complementation assay for identification and purification of specific components.